SUMMARY: Intestinal barrier function is compromised in infectious and immune-mediated intestinal diseases. This program, now completing its third funding cycle, has focused on the mechanisms and impact of intestinal epithelial barrier dysfunction. In previous cycles we defined epithelial myosin light chain kinase (MLCK) as a critical regulator of tight junction barrier function in response to inflammatory stimuli. This work has been replicated widely and extended to other systems. We went on to show that MLCK-dependent increases in permeability promote progression of inflammatory bowel disease and graft-versus-host disease. We also defined signaling events downstream of MLCK activation and found that while the barrier is regulated by the immune system, MLCK-dependent increases in tight junction permeability are also able to regulate mucosal immunity that triggers increases in claudin-2 expression. This led to our co-discovery of the pore and leak pathways of trans-tight junction flux; a model that is now widely accepted. In the current cycle we focused on the pore and leak pathways and found that, in the context of infectious disease, increased tight junction permeability as a result of claudin-2 expression is beneficial and promotes pathogen clearance. In addition, we identified a specific MLCK splice variant, MLCK1, as a critical regulator of tight junction permeability. In addition to activating MLCK transcription and enzymatic activity, we found that inflammatory stimuli cause MLCK1 to be recruited to the perijunctional actomyosin ring. We identified a specific domain, immunoglobulin- cell adhesion molecule domain 3 (IgCAM3) as being required for MLCK1 recruitment and sufficient to act as a dominant negative to block recruitment. We went on to solve the IgCAM3 crystal structure, identify a potential drug-binding pocket that was conserved between human and mouse IgCAM3 but absent in other MLCK IgCAMs, and perform an in silico screen of a NCI library of ~140,000 drug-like molecules. We identified one, termed Divertin, that prevents MLCK1 recruitment without inhibiting epithelial or smooth muscle MLCK enzymatic function. By blocking MLCK1 recruitment to the perijunctional actomyosin ring, Divertin prevents MLCK1 from phosphorylating myosin II regulatory light chain at that site. This, in turn, blocks inflammation- induced barrier loss in vitro, in vivo (mice), and ex vivo (human intestinal biopsies). Divertin attenuated all features of experimental immune-mediated inflammatory bowel disease (mice), including barrier loss, immune activation, and mortality. The aim of this proposal is to identify the intracellular protein interactions modified by Divertin and define the molecular mechanisms of MLCK1 recruitment. The studies described will characterize MLCK1 binding partners we have already discovered as well as new binding partners identified through cutting-edge proteomic approaches. At completion, these studies will have defined the MLCK1 interactome and mechanisms by which recruitment is regulated as well as the potential benefits and risks of inhibiting MLCK1 recruitment. These advances will enable future development of Divertin-like agents for human use.